[go: up one dir, main page]

EP2400974B1 - Hydrogels de co-polypeptide à double bloc synthétique pour une utilisation dans le système nerveux central - Google Patents

Hydrogels de co-polypeptide à double bloc synthétique pour une utilisation dans le système nerveux central Download PDF

Info

Publication number
EP2400974B1
EP2400974B1 EP10744302.0A EP10744302A EP2400974B1 EP 2400974 B1 EP2400974 B1 EP 2400974B1 EP 10744302 A EP10744302 A EP 10744302A EP 2400974 B1 EP2400974 B1 EP 2400974B1
Authority
EP
European Patent Office
Prior art keywords
dch
poly
composition
peptide
vivo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Not-in-force
Application number
EP10744302.0A
Other languages
German (de)
English (en)
Other versions
EP2400974A4 (fr
EP2400974A2 (fr
Inventor
Timothy J. Deming
Michael V. Sofroniew
Chu-Ya Yang
Bingbing Song
Yan AO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP2400974A2 publication Critical patent/EP2400974A2/fr
Publication of EP2400974A4 publication Critical patent/EP2400974A4/fr
Application granted granted Critical
Publication of EP2400974B1 publication Critical patent/EP2400974B1/fr
Not-in-force legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/25—Peptides having up to 20 amino acids in a defined sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A61L2300/254—Enzymes, proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/44—Radioisotopes, radionuclides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/442—Colorants, dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
    • A61L2300/624—Nanocapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

  • bioactive molecules with therapeutic potential for the central nervous system exert wide-ranging activities that can lead to unwanted side effects if they are delivered systemically.
  • a number of potential therapies for neurological conditions have failed in clinical trials for this reason.
  • Site-specific delivery of potential therapies is increasingly recognized as an important goal for many neurological conditions, and biomaterial vehicles represent a promising means of achieving this goal.
  • suitable vehicles are not presently available for the restricted targeting of growth factors or other diffusible molecules, or the placement of extracellular matrices for axon growth or cell migration.
  • neural stem/progenitor cell grafts may hold promise for replacing lost cells in certain neurological conditions, but most current grafting procedures are regarded as resulting in suboptimal survival and differentiation of cells.
  • Biomaterial vehicles may be able to improve grafting efficiency by providing grafted cells with support matrix and molecular substrates that help them overcome the shock of the grafting procedure and integrate and differentiate better in to host tissue.
  • Injectable biomaterials represent a rapidly advancing new area for delivery of therapeutic molecules.
  • the U.S. F.D.A. has already approved certain biomaterials for slow release, long-term systemic drug delivery, while others are currently undergoing clinical trials
  • the basic precedent of using biomaterials for drug delivery in humans has thus been set, but no materials are currently approved for delivery into the CNS.
  • Hydrogels are a class of materials that have significant promise for use in soft tissue and bone engineering ( Lee et al. (2001) Hydrogels for tissue engineering, Chemical Reviews 101, 1869-1879 ).
  • An important feature of hydrogels that makes them attractive for these applications is their well hydrated, porous structure that can mimic natural extracellular matricies ( Peppas et al. (2000) Physicochemical foundations and structural design of hydrogels in medicine and biology, Annual Review of Biomedical Engineering 2, 9-29 ). To replace natural materials, however, many structural and functional features must be built into synthetic hydrogels.
  • Desirable features include: biocompatability; degradability to allow cell ingrowth; injectable yet also fast setting in the wound site; mechanical properties that can be tuned for different uses; control over cell adhesion to the hydrogel matrix; and tunable sustained release of growth factors and other biologically active agents.
  • biocompatability There are many examples where some, or even most of these features have been incorporated into hydrogels. However, in many cases, hydrogel synthesis and formation becomes very complicated, which limits the practicality of such materials. More importantly, the complexity of these systems, combined with limited means for adjustment of molecular parameters, leads to the inability to independently adjust most of the features. For example, it would be advantageous to be able to adjust scaffold rigidity while maintaining a constant hydrogel mesh size.
  • Such a system would allow one to directly measure the effects of scaffold rigidity on cell proliferation. Also, since hydrogel degradation is commonly accomplished using degradable crosslinkers (e.g. in PEG based hydrogels), it can be difficult to adjust degradation rate without also altering crosslink density, and hence initial gel mechanical properties. It would be desirable to have a hydrogel system where many of these desirable adjustable features (e.g. gel strength, gel density, adhesive capability, degradation rate, growth factor release rate) could be controlled independently so that, e.g., meaningful evaluations of their roles in tissue regeneration could be systematically evaluated. Currently, in many systems it is difficult to identify the most important gel characteristics, since many features are adjusted simultaneously.
  • these desirable adjustable features e.g. gel strength, gel density, adhesive capability, degradation rate, growth factor release rate
  • hydrogel technology utilizes both naturally-derived macromolecules and synthetic polymers.
  • hydrogels prepared from natural polymers possess desired biological signalling capability but may lack desired material properties, e.g. low sample rigidity, and may also be problematic due to immunogenicity and pathogen transmission issues.
  • synthetic polymers can be engineered for desired material properties but may display limited cytocompatibility.
  • One approach to increase the cytocompatibility of synthetic polymers is to incorporate peptide epitopes, for example RGD motifs.
  • RGD motifs peptide epitopes
  • these motifs into preformed polymers in a regiospecifically controlled manner is extremely difficult.
  • these scaffolds are structurally homogeneous (not porous) on the microscale due to their underlying molecular network structure, which can limit cell proliferation.
  • hydrogel rigidity seemingly precludes any viable processability in preformed scaffolds. For example, one may wish to form a rigid tissue engineering construct in vitro but subsequently inject it into a host for tissue regeneration. Injection is not possible in a permanently crosslinked, rigid network.
  • hydrogels for tissue engineering applications severely prohibits the use of materials with a large degree of adjustability in their properties.
  • the polymers used in most synthetic hydrogels simply do not contain enough functionality to allow tuning of degradability, adhesion or gel strength without compromising other necessary properties.
  • DCH Amphiphilic diblock copolypeptide hydrogels
  • the present inventors have previously used a combination of chemical synthesis and structural characterization to establish an understanding of DCH structure-property relationships that allows a high level of control over gel strength, gel porosity, gel functionality and media stability; and many of these properties can be adjusted independently of each other ( Nowak et al. (2002) Rapidly recovering hydrogel scaffolds from self-assembling diblock copolypeptide amphiphiles, Nature 417, 424-428 ; Breedveld et al. (2004) Rheology of block copolypeptide solutions: hydrogels with tunable properties, Macromolecules, 3943-395 )).
  • DCH are physically associated gels that can be deformed and thinned by stress and injected through small-bore cannulae, after which they rapidly re-assemble into rigid gel networks (Nowak et al. (2002) ( supra )), These properties provide DCH with the potential for facile and minimally invasive delivery in vivo, DCH form elastic gels with fibril-like nanostructures and porous microstructures theoretically suitable for integration with host cells (Nowak et al. (2002), ( supra ); Deming (2005), ( supra ).
  • DCH could be generated that are suitable for administration to the CNS, or whether such DCH could serve as depots for biologically active materials or act as scaffolds to support cell migration in the CNS.
  • the present inventors identify formulations of synthetic amphiphilic diblock copolypeptide hydrogels (DCH) that are suitable for use in the central nervous system (CNS), demonstrate methods of making these DHC, and show that the DCH can be used to deliver molecules to specific sites in the CNS (e.g., into cells in the brain or spinal cord).
  • Molecules of interest such as diagnostic or therapeutic molecules, can be delivered by releasing diffusible biologically active molecules that are mixed with (e.g., dissolved, dispersed, or suspended in) the gel matrix, or that have been conjugated in a reversible or non-reversible fashion to the polypeptide backbone of the DCH.
  • DCH that have been decorated with suitable guides which provide migratory cues are also shown to function as extracellular scaffolds, e.g. for the migration of cells, growth of nerve fibers, blood vessel formation, or the like.
  • the inventors evaluate a variety of block copolymer hydrogel (including diblock copolymer hydrogel, or DCH) formulations, comparing their physical properties in vitro with their properties in vivo after injection into mouse forebrain, and identify some formulations that are particularly suitable for use in the CNS. These formulations are shown to exhibit good biocompatibility with little or no detectable toxicity in the CNS, and can be tuned to exhibit a range of desirable physical properties that make them useful for potential applications in the CNS, as depots or as scaffolds.
  • block copolymer hydrogel including diblock copolymer hydrogel, or DCH
  • the inventors also develop methods for conjugating biologically active molecules (such as peptides or proteins) to DCHs, in a manner such that the biologically active molecules are released in a controlled fashion.
  • biologically active molecules such as peptides or proteins
  • the inventors demonstrate, as a representative example, that amphiphilic DCHs can serve as depots for chondroitinase ABC (chABC), and that bioactive chABC can be delivered and released following injection into brain tissue in vivo.
  • migration guidance cues such as laminin or bioactive peptides thereof (e.g. IKVAV (SEQ ID NO:1) or YIGSR (SEQ ID NO:2)
  • DHCs can be loaded with nanocarriers (e.g. vesicles or emulsions) that comprise cargo (e.g., nucleic acids, oligonucleotides (such as siRNAs or microRNAs), peptides, oligopeptides, proteins (including enzymes), carbohydrates, viruses, cells, etc.) which can be delivered to and released in a sustained fashion to cells, in vitro and in vivo.
  • cargo e.g., nucleic acids, oligonucleotides (such as siRNAs or microRNAs), peptides, oligopeptides, proteins (including enzymes), carbohydrates, viruses, cells, etc.
  • Such nanocarriers are particularly useful for entrapping and delivering molecules for which it is desirable to prevent degradation.
  • DCH of the invention include, e.g., that they are safe, efficacious, have the potential for GMP manufacture, and their production can readily be scaled up to levels required for translational purposes.
  • DCH can easily and reproducibly be produced in large quantities of high purity with complete removal of chemicals employed during synthesis; and the samples are free from pyrogens or biological contaminants and are easily sterilized by autoclave.
  • Such easy to use and well-understood delivery systems can achieve site-specific delivery of bioactive molecules and cells into the brain and spinal cord over controlled periods of time, and can be used in therapeutic strategies for a wide variety of CNS disorders.
  • the unprecedented versatility of these block copolypeptide hydrogen systems provide multiple molecule adjustments to tune different material properties, such as gel strength or porosity.
  • One aspect of the invention is a composition suitable for administration to the central nervous system (CNS), which comprises a block copolypeptide hydrogel (which is made of a polypeptide backbone in association with an aqueous material, forming a hydrogel).
  • CNS central nervous system
  • a block copolypeptide hydrogel which is made of a polypeptide backbone in association with an aqueous material, forming a hydrogel.
  • DCH di block copolypeptide hydrogel
  • the DCH comprises a biologically active material (sometimes referred to herein as a bioactive material, a bioactive molecule, or a biomolecule) that is (a) mixed with (e.g., suspended in, dispersed in, or dissolved in) the aqueous pores or the hydrogel matrix portion of the DCH, and/or (b) attached (e.g., covalently attached) to the polypeptide backbone of the hydrogel.
  • a biologically active material sometimes referred to herein as a bioactive material, a bioactive molecule, or a biomolecule
  • a biologically active material (sometimes referred to herein as a bioactive material, a bioactive molecule, or a biomolecule) that is (a) mixed with (e.g., suspended in, dispersed in, or dissolved in) the aqueous pores or the hydrogel matrix portion of the DCH, and/or (b) attached (e.g., covalently attached) to the polypeptide backbone of the hydrogel.
  • a composition of the invention can be used as a depot to deliver a diagnostic or therapeutic agent intracellularly to the brain or spinal cord, or it can serve as a scaffold for, e.g., cell migration, organogenesis, nerve fiber growth and/or blood vessel formation.
  • the biologically active material is a diagnostic or therapeutic agent that is mixed with the DCH.
  • the DCH acts as a depot for that material.
  • the biologically active material is covalently bound to the polypeptide backbone of a DCH, reversibly or non-reversibly.
  • the biologically active material can be, e.g., a peptide, polypeptide, sugar, oligosaccharide, polysaccharide, glycoprotein, or oligonucleotide.
  • the biologically active material can be, e.g., a therapeutic agent or a diagnostic agent (e.g., an MRI contrast agent (such as Gd3+ ions, which can be used for MRI imaging), a radioisotope or a fluorescent probe).
  • the DCH can act as a depot for the covalently attached material.
  • the biologically active material is a peptide or polypeptide which is covalently attached to the polypeptide backbone of the DCH; and the peptide or polypeptide functions as a migration guidance cue.
  • peptides or polypeptides include a laminin or a biologically active peptide thereof, such as, e.g., IKVAV (SEQ ID NO:1), or YIGSR (SEQ ID NO:2).
  • the biologically active material is a diagnostic or therapeutic agent that is entrapped in a nanocarrier (e.g., a submicron particle, such as a vesicle, an emulsion, or a double emulsion droplet) which, in turn, is mixed with a block copolypeptide hydrogel of the invention.
  • a nanocarrier e.g., a submicron particle, such as a vesicle, an emulsion, or a double emulsion droplet
  • a double emulsion droplet comprises a mixture of hydrophilic and hydrophobic regions and, itself, can be taken up by a cell.
  • the block copolypeptide can be a diblock copolypeptide, which may comprise or consist essentially of, e.g .,
  • Another aspect of the invention is a method for making a composition of claim 1, comprising covalently attaching a peptide or protein of interest to the polypeptide backbone, by
  • Another aspect of the invention is the compound for use in introducing biologically active chABC into a brain (e.g., a forebrain) in vivo, comprising injecting a composition of the invention in which chABC is mixed with the DCH or is covalently attached to the polypeptide backbone into the brain (e.g., forebrain).
  • a biologically active chABC into a brain (e.g., a forebrain) in vivo, comprising injecting a composition of the invention in which chABC is mixed with the DCH or is covalently attached to the polypeptide backbone into the brain (e.g., forebrain).
  • Another aspect of the invention is a method for providing a scaffold for cell migration in the central nervous system (CNS), comprising functionalizing a diblock copolypepide hydrogel of the invention with a protein or peptide that provides a migration guidance cue (e.g. a laminin or a biologically active peptide thereof).
  • a migration guidance cue e.g. a laminin or a biologically active peptide thereof.
  • Another aspect of the invention is the compound for use in stimulating cell migration, or angiogenesis, or for use in stimulating the in-growth of nerve fibers, in a brain, comprising introducing into the brain a scaffold that is produced as above.
  • a method can, e.g., enhance axonal plasticity and/or improve functional recovery after peripheral nerve repair following spinal cord injury.
  • kits comprising: (a) a block copolypeptide of claim 1, which has been lyophilized, and, an aqueous solution comprising a therapeutic agent, an imaging agent, or a submicron particle encapsulating an agent of interest, with which the lyophilized block copolypeptide can be reconstituted; or (b) a block copolypeptide of claim 1, which been chemically functionalized with a biomolecule of interest and which has been lyophilized, and, an aqueous solution with which the functionalized, lyophilized block copolypeptide can be reconstituted.
  • CNS central nervous system
  • brain and spinal cord.
  • a “biologically active material” (sometimes referred to herein as a bioactive molecule or agent), as used herein, is a material that exhibits a measurable amount of a biological activity.
  • a diagnostic agent can exhibit the ability to stain or image a region of interest; a therapeutic agent can inhibit a symptom of a pathological condition; and a migration guidance cue can provide a signal for a cell to migrate.
  • a biologically active peptide from a protein e.g., a peptide sequence from a laminin
  • retains at least one biological activity of the full-length protein e.g., the ability to provide a migration guidance cue.
  • a “migration guidance cue” is meant the ability to direct the migration of cells in space or tissue.
  • compositions are compatible with (e.g., exhibits minimal toxicity, gliosis, or inflammation) to the cells/tissues in the CNS to which it is administered.
  • a composition that is suitable for administration to the CNS may also exhibit one or more of the following advantageous properties: (a) the rheological property of thinning while under shear (e.g., extrusion through a small gauge needle), for ease of processing, but then is able to return immediately via self-assembly after cessation of shear; (b) the completely peptidic nature of the molecular blocks provides an opportunity for facile incorporation of specific secondary structures (e.g., hydrophobe conformation) to manipulate gelatin conformation, by changing which amino acids are in the diblock, relative block lengths, etc.); and (c) degradation over time in vivo to release the depot.
  • specific secondary structures e.g., hydrophobe conformation
  • minimally cytotoxic maintenance of cell viability as compared to a suitable control, e.g., as determined using the assays described in the Examples below.
  • a DCH includes multiple DCH, e.g. 2, 3, 4, 5 or more DCH, which can be the same or different.
  • a "subject,” as used herein, includes any animal that contains an CNS. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates, including human patients, are included.
  • a “therapeutic effect,” as used herein, refers to a therapeutic response, such as the amelioration of a symptom associated with a pathological condition or disease, particularly a neurological condition of the CNS.
  • a therapeutic effect can, for example, comprise the prevention, reduction, treatment or elimination of a particular condition.
  • an "effective" amount refers to an amount that can bring about at least a detectable (measurable) amount of a desired response of interest.
  • a “therapeutically effective amount” refers to an amount that can bring about at least a detectable therapeutic effect.
  • amphiphilic block copolypeptides of the present invention comprise at least two distinct polypeptide segments (sometimes referred to herein as blocks, domains, or moieties), which are covalently linked: (1) one or more hydrophilic moieties, sometimes referred to herein as water-soluble, soluble, polar, or charged (anionic or cationic) moieties, and (2) one or more hydrophobic moieties, sometimes referred to herein as a water-insoluble, insoluble, lipophilic, or non-polar moieties.
  • one or more hydrophilic moieties sometimes referred to herein as water-soluble, soluble, polar, or charged (anionic or cationic) moieties
  • hydrophobic moieties sometimes referred to herein as a water-insoluble, insoluble, lipophilic, or non-polar moieties.
  • DCH diblock copolypeptide hydrogel
  • trimer copolypeptide hydrogel
  • Additional segments can be present, e.g., 4, 5 or more segments.
  • the hydrophilic block(s) of the copolypeptides comprise amino acids with charged side-chains, with the amino acids being, for example: glutamic acid (E), aspartic acid (D), arginine (R), homoarginine (R H ), histidine (H), lysine (K), or ornithine (O).
  • the amino acids can be naturally occurring amino acids or D-isomers. Racemic mixtures of D- and L-amino acids are included.
  • the hydrophilic block is made up of identical amino acids, to form a homopolypeptidic domain.
  • the hydrophilic domain is made up of two or more different amino acids, which can be mixed in any ratio, to form a heteropolypeptidic domain.
  • a hydrophilic block of the invention contains only polar amino acids. However, in embodiments of the invention, a few non-polar amino acids may also be present, but they must be present in less than about 10% of the total number of amino acids.
  • the hydrophilic block comprises:
  • the hydrophobic block(s) comprise amino acids with nonionic (non-polar) side-chains, with the amino acids being, for example: phenylalanine (F), leucine (L), valine (V), isoleucine (I), alanine (A), serine (S), threonine (T) or glutamine (Q).
  • the amino acids can be naturally occurring amino acids or D-isomers. Racemic mixtures of D- and L-amino acids are included.
  • the hydrophobic block is made up of identical amino acids, to form a homopolypeptidic domain.
  • the hydrophobic domain is made up of two or more different amino acids, which can be mixed in any ratio, to form a heteropolypeptidic domain.
  • a hydrophobic block of the invention contains only non-polar amino acids. However, in embodiments of the invention, a few polar amino acids may also be present, but they must be present in less than about 10% of the total number of amino acids.
  • the hydrophobic block comprises:
  • the length of a copolypeptide chain of the invention is between 100 - 600 amino acid residues, e.g., about 100, 200, 300, 400, 500 or 600 residues. In one embodiment, which is exemplified herein, the polypeptide chain is about 200 amino acids in length.
  • the ratio of hydrophilic to hydrophobic amino acids in a copolypeptide chain of the invention can range from about 50-95 mole %.
  • the hydrophobic block in a diblock copolypeptide of 200 amino acids, can be 100-190 amino acids, and the hydrophilic block, 100-10 amino acids, respectively.
  • the block copolypeptide is the diblock copolypeptide, K 180 L 20 (SEQ ID NO:8).
  • DCHs of the invention are made by allowing block copolymers to self-assemble in an aqueous solution under suitable conditions to form rigid hydrogels.
  • the pH of the aqueous medium is about pH 4-10, e.g., about pH 6-8; the temperature is about 4°C-100°C; and the concentration of the polypeptide is very low (e.g., between about 0.01%-30%, or between about 0.1%-5% (wt% (g/vol), or weight/volume)).
  • the amount of copolymer present in the mixture may vary.
  • the amount of copolymer present in the mixture ranges from 0.1% weight/volume (w/v) to 5%, such as from about 0.5 to about 3% and including from about 1 to about 2%.
  • the nature of the aqueous medium used to generate DCHs of the invention is a function of what biologically active agents, are to be encapsulated in the DCHs.
  • the concentration of a bioactive agent may vary. In certain embodiments the concentration of active agent present in the mixture ranges from about 1 nM to about 100 mM, such as from about 1 ⁇ M to about 100 ⁇ M. This concentration will also depend on the potency of the active agent.
  • acceptable aqueous media will be evident to a skilled worker. These include, e.g., sterilized and/or dionized water, buffers (such as PBS), physiological saline, artificial cerebral spinal fluid, or cell culture media (DMEM).
  • a block copolypeptide hydrogel of the invention has a heterogeneous microstructure with distinct domains of hydrogel matrix (on the nanoscale) and aqueous (e.g. pure water) pores (on the microscale), as a porous, interconnected membranous network of assembled polypeptides.
  • the elastic gels formed by the netwok of polypeptide chains are suitable for integration into host cells.
  • Figure 1 illustrates the structure of DCH of the invention.
  • polypeptide backbone The polypeptide component of a DCH is sometimes referred to herein as the "polypeptide backbone.”
  • DCH can be deformed by stress, such as extrusion through a small gauge needle, allowing them to be injected as liquids that rapidly re-assemble into gels.
  • DCH are synthesized using chemical polymerization, thus obtainable in pure form with no biological contaminants, and are easily manipulated to achieve different properties for potential applications with different requirements, while retaining basic attributes.
  • the components and properties of DCH can be varied intentionally to achieve different attributes when injected in vivo by, for example, varying the amino acid composition of the copolypeptide blocks, the ratios of hydrophobic and hydrophilic components of the amphiphiles, and/or the concentration of the DCH formulations, which affect gel strengths of the DCH preparations. By varying several factors, one can enable either easy diffusion through tissue, or the provision of three-dimensional (3D) structure to replace lost tissue or bridge cavities.
  • the polypeptide backbone of DCH allows straightforward incorporation of peptidic functionality that can, e.g., provide a depot of a diagnostic or therapeutic agent which can subsequently be released and delivered into a cell, or which can impart cell adhesion, molecular signaling or enzymatic degradability, either by direct incorporation of desired amino acids into the primary polypeptide chain, or by attaching sequence specific peptides at defined locations on the chains so that bioactive groups can be linked to the polypeptide chains with controlled density and spatial resolution.
  • Typical methods for covalently attaching peptides or proteins to a polypeptide backbone of a DCH are described in Example II.
  • amine-coupling or thiol-ene coupling can be used to generate irreversible bonds.
  • 4-allyloxy-4-oxo-butanoic acid has an alkene group on one end that can be used for thioliene coupling to thiolated proteins, and its other end is a carboxylic group that can be coupled to the N ⁇ -amine on the side chain of lysine in DCH.
  • ester group in the middle of the linker that should hydrolyze slowly over time under physiological conditions.
  • Other cleavable cross-linkers will be evident to a skilled worker. These include, e.g., disulfide bonds which will cleave upon reduction.
  • Other molecules that can be attached to a polypeptide backbone e.g., MRI contrast agents, radioisotopes or fluorescent probes, or other molecules described herein
  • can be attached using identical chemistry i.e. either amide bond formation if the molecule has an amine or carboxylate group (non-cleavable), or using the thiol-ene chemistry by attaching either a thiol or alkene to the molecule of interest).
  • a variety of types of cargo/payload can be loaded into DCH of the invention.
  • the loading can be performed in a non-covalent manner such that the agent is dissolved or dispersed within the polar medium, is associated with the DCH through a non-covalent relationship with the matrix of the hydrogel, is embedded in the hydrogel, or combinations thereof.
  • one or more of the self-assembling block copolypeptides of a DCH may be covalently modified with an agent of interest.
  • the agent may be attached to a residue of the polypeptide backbone through a biodegradable bond, such as a disulfide or ester, which bond may include a linker or spacer on either or both sides.
  • the DCH may include both covalent and non-covalently attached agents of interest, as well as single and multiple different payloads, depending on a given end use.
  • Some types of cargo to be delivered are hydrophilic, and thus can be readily loaded in a non-covalent manner into the polar interior of a DCH.
  • These water-soluble agents include, e.g., peptides or proteins (such as growth factors, cytokines, function-blocking hybrid proteins, and certain enzymes), peptidomimetics, nucleic acids, oligonucleotides (such as siRNAs, antisense DNA molecules, microRNAs, etc), nucleotides, nucleosides, carbohydrates, and a variety of diagnostic agents, and therapeutic or prophylactic agents, including anti-bacterial agents, anti-viral agents, and cognition enhancers.
  • Polar cargo such as, e.g., steroids, sterols, dyes such as 5-dodecanoylaminofluorescein, and certain drugs, can also be introduced into DCH of the invention.
  • a hydrophobic cargo if interest can be attached covalently or admixed with a suitable amphiphilic surfactant for its dispersion or containment in a polar medium suitable for encapsulation into a DCH of the invention.
  • amphiphilic surfactants for this purpose include, for instance, polyethoxylated fatty acids, such as the PEG-fatty acid monoesters and diesters of lauric acid, oleic acid, and stearic acid (as well as PEG-glycerol fatty acid esters of lauric acid, oleic acid, and stearic acid), amphiphilic transesterification products of oils and alcohols, sterols and sterol derivatives, oil-soluble vitamins.
  • polyethoxylated fatty acids such as the PEG-fatty acid monoesters and diesters of lauric acid, oleic acid, and stearic acid
  • PEG-glycerol fatty acid esters of lauric acid, oleic acid, and stearic acid amphiphilic transesterification products of oils and alcohols, sterols and sterol derivatives, oil-soluble vitamins.
  • vitamins A, D, E, K, etc. such as vitamins A, D, E, K, etc., polyglycerol esters of fatty acids as well as mixtures of surfactants such as propylene glycol fatty acid esters and glycerol fatty acid esters, amphiphilic esters of sugars such as sucrose monopalmitate and sucrose monolaurate, sucrose monostearate, sucrose distearate, amphiphilic esters of lower alcohols (C2 to C4) and fatty acids (C8 to C8) and the like.
  • surfactants such as propylene glycol fatty acid esters and glycerol fatty acid esters
  • amphiphilic esters of sugars such as sucrose monopalmitate and sucrose monolaurate, sucrose monostearate, sucrose distearate, amphiphilic esters of lower alcohols (C2 to C4) and fatty acids (C8 to C8) and the like.
  • Active biological agents can also be loaded into a DCH of the invention by encapsulating them first in a nanocarrier, such as a vesicle, emulsion, or a double emulsion droplet.
  • a nanocarrier such as a vesicle, emulsion, or a double emulsion droplet.
  • compositions of the invention can be used in a variety of ways, in vitro or in vivo, including, e.g., research applications, the formation of scaffolds which support cell migration, or restricted targeting of diffusible molecules for the diagnosis of neurological conditions (e.g., imaging technology) or for the delivery of therapeutic agents to treat neurological conditions.
  • DCH of the invention can deliver agents that can facilitate research applications, such as cell-based assays (e.g., intracellular delivery of ion indicators, reactive dyes and chemicals, or primary or secondary detection and/or quantitation components).
  • cell-based assays e.g., intracellular delivery of ion indicators, reactive dyes and chemicals, or primary or secondary detection and/or quantitation components.
  • DCH of the invention can deliver agents to the CNS for diagnostic purposes, such as imaging and contrast agents.
  • Suitable diagnostic agents include, e.g., a detectable label or a reporter ligand, which includes both active and passive reporter ligands such as a component of a fluorescence resonance energy transfer (FRET) detection system, spin-trap agents, quantum dots, chelated agents, contrast agents, dyes, radiolabels, peptides, nucleic acids, antibodies, antibody fragments and the like.
  • FRET fluorescence resonance energy transfer
  • DCH loaded with diagnostic agents can be used in connection with a variety of detection and imaging modalities, such as those involving standard analytic and/or separation-based detection modalities (e.g., chromatography, Enzyme-Linked ImmunoSorbent Assays (ELISA) etc.), as well as those based on less invasive modalities such as gamma-scintigraphy, magnetic resonance imaging and computed tomography).
  • detection and imaging modalities such as those involving standard analytic and/or separation-based detection modalities (e.g., chromatography, Enzyme-Linked ImmunoSorbent Assays (ELISA) etc.), as well as those based on less invasive modalities such as gamma-scintigraphy, magnetic resonance imaging and computed tomography).
  • DCH can be loaded with chelated or bifunctional chelated agents (e.g., covalent linkage group coupled to a targeting moiety such as an antibody, antibody fragment, peptide or hormone and a chelating group for the metal) and used (depending on the particular agent selected and modality of administration) for, e.g., myelography (radiological study of the spinal cord); CAT scan (Computerized Axial Tomography as a method of resolution of a series of x-ray pictures into a "cross-section" of the body or part of the body, such as the brain, in which a contrast agent may be employed), NMR scan or MRI (Magnetic Resonance Imaging as a computerized method of resolution of a series of radio-frequency scans of tissues into a "cross-section" of the body or body part, such as the brain, which visualizes in a tissue-specific manner the composition of areas rather than density as in the CAT scan).
  • chelated or bifunctional chelated agents e.g., covalent
  • the diagnostic agent employs technecium.
  • This agent is used in 85% of all medical diagnostic scans, easily forms metal-electron donor complexes or chelates in the presence of a reducing agent, such as electronegative chelating groups illustrated by SH thiols, CO 2 -carboxylates, NH amines, PO 4 -phosphate, CNOH oximes, OH hydroxyls, P phosphines, and NC isonitriles, exhibits good properties for imaging with a gamma camera, and possesses a short half-life of 6 hours that is adequate to synthesize chelate, determine purity, administer and image with a minimum radiation exposure.
  • a reducing agent such as electronegative chelating groups illustrated by SH thiols, CO 2 -carboxylates, NH amines, PO 4 -phosphate, CNOH oximes, OH hydroxyls, P phosphines, and NC isonitriles
  • Illustrative chelated agents include technecium tagged agents such as, e.g., technecium biscisate (which can be used, e.g., for imaging to determine brain perfusion in stroke and lesion determination); technecium exametazine (a brain imaging agent to determine brain death in life support patients, localize seizure foci, dementia, strokes); technecium gluceptate (e.g., for radiolabeling of monoclonal antibodies); and technecium pentetate (e.g., imaging of the brain for brain tumors and death).
  • technecium tagged agents such as, e.g., technecium biscisate (which can be used, e.g., for imaging to determine brain perfusion in stroke and lesion determination); technecium exametazine (a brain imaging agent to determine brain death in life support patients, localize seizure foci, dementia, strokes); technecium gluceptate
  • radiolabel generators in addition to technecium include complexes of strontium-yttrium, zinc-copper, germanium-gallium, strontium-rubidium, gallium citrate, 18F-2-fluoro-2-deoxy-D-glucose (e.g., PET scanning (positron emission tomography) for determining metabolic rate in the brain, and cancer management (neoplasms have a high glycolytic rate) etc.), iodine radiolabels (e.g., iobenguane sulfate 131 I for imaging and locating functional neuroblastomas), indium radiolabels (e.g., utilized to radiolabel monoclonal antibodies and peptides via bifunctional chelating agents; such as indium chloride which behaves similar to Fe 3 for imaging of tumors); indium satumomabpendetide for labeling of monoclonal antibodies; and indium oxine (8-hydroxyquinoline) for replacing gallium radiol
  • Additional diagnostic agents include radiological contrast agents such as the iodine based compounds (e.g., diatrizoate megllumine, distrizoate sodium, iopanoic acid, tryopanoate sodium, ipdoate sodium, iothalamate meglumine, iodipamide meglumine, iohexol, iopamidol, ioversol, iodixanol, isosulfan blue, pentetreotide), MRI contrast agents (e.g., gadolinium chelated compounds such as gadopentetate dimeglumin, gadoteridol, ferummoxsil, ferumoxides, masngofodipir trisodium), and ultrasound contrast agents (e.g., perflexane-n-perfluorohexane gas, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)).
  • DCH of the invention can be used as depots for intracellular delivery (e.g., sustained molecular release) of therapeutic (including prophylactic) agents.
  • therapeutic agents include, e.g., growth factors, cytokines, function-blocking hybrid proteins, inhibitors, certain enzymes, oligonucleotides (such as siRNAs, antisense DNA molecules, microRNAs, etc), anti-bacterial agents, anti-viral agents, cognition enhancers, etc.
  • Example II describes the loading and delivery of one representative therapeutic agent, chondroitinase ABC (ch ABC), in a DCH of the invention.
  • DCH of the invention can be used to provide extracellular matrices (scaffolds) to support and guide cell migration in the CNS.
  • a scaffold can bridge cell and nerve fiber migration across scar tissue or to fill wound cavities; can support axon growth; or can support grafted cells.
  • Neural stem/progenitor cell grafts hold promise for replacing lost cells in certain neurological conditions (e.g. neurodegenerative diseases, such as multiple sclerosis, or stroke), but most current grafting procedures are regarded as resulting in suboptimal survival and differentiation of cells.
  • Compositions of the invention can improve grafting efficiency by providing grafted cells with support matrix and molecular substrates that help them overcome the shock of the grafting procedure and integrate and differentiate better in to host tissue.
  • polypeptide chains of the DCH are functionalized with appropriate migration guidance cues, examples of which will be evident to a skilled worker.
  • appropriate migration guidance cues include, e.g ., the alpha chain of laminins or active peptides thereof, such as ile-lys-val-ala-val, or IKVAV (SEQ ID NO:1); tyr-ile-gly-ser-arg, or YIGSR (SEQ ID NO:2); ser- ile-lys-val-ala-val (SIKVAV) (SEQ ID NO:3); RNIAEIIKDI (SEQ ID NO:4), and SRARKQAASIKVAVSADR (SEQ ID NO:5).
  • Other suitable migration cues will be evident to a skilled worker. These include, e.g., whole growth factors such as BNDF, NT3, or NGF.
  • compositions discussed herein can be formulated into various compositions, for use in diagnostic or therapeutic treatment methods.
  • the compositions e.g. pharmaceutical compositions
  • a pharmaceutical composition of the invention comprises an effective amount (e.g., a pharmaceutically effective amount) of a composition of the invention.
  • a composition of the invention can be formulated as a pharmaceutical composition, which comprises a composition of the invention and pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e ., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • pharmaceutically acceptable carriers and other components of pharmaceutical compositions see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990 .
  • suitable pharmaceutical carriers include, e.g., water (including sterile and/or deionized water), suitable buffers (such as PBS), physiological saline, cell culture medium (such as DMEM), artificial cerebral spinal fluid, or the like.
  • a pharmaceutical composition or kit of the invention can contain other pharmaceuticals, in addition to the compositions of the invention.
  • the other agent(s) can be administered at any suitable time during the treatment of the patient, either concurrently or sequentially.
  • compositions of the present invention will depend, in part, upon the particular agent that is employed, and the chosen route of administration. Accordingly, there is a wide variety of suitable formulations of compositions of the present invention.
  • Formulations which are suitable for topical administration directly in the CNS include, e.g., suitable liquid carriers, or creams, emulsions, suspensions, solutions, gels, creams, pastes, foams, lubricants, or sprays. Topical administration in the CNS is possible when the CNS is opened by wound or during a surgery.
  • compositions of the invention can be in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for animal (e.g. human) subjects, each unit containing a predetermined quantity of an agent of the invention, alone or in combination with other therapeutic agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.
  • One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired effective amount or effective concentration of the agent in the individual patient.
  • compositions of the invention can be administered with a catheter or a needle (syringe).
  • a composition of the invention can be administered directly to the exposed tissue, e.g. by topical administration.
  • the dose of a composition of the invention, administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect at least a detectable amount of a diagnostic or therapeutic response in the individual over a reasonable time frame.
  • the dose used to achieve a desired effect will be determined by a variety of factors, including the potency of the particular agent being administered, the pharmacodynamics associated with the agent in the host, the severity of the disease state of infected individuals, other medications being administered to the subject, etc.
  • the size of the dose also will be determined by the existence of any adverse side effects that may accompany the particular agent, or composition thereof, employed. It is generally desirable, whenever possible, to keep adverse side effects to a minimum.
  • a suitable dose of a composition of the invention to be administered to the brain is generally determined as a function of the volume of the brain. For example, for a mouse, whose brain has a volume of about 1-2 cc, one generally administers about 1 ⁇ L- 10 ⁇ L of a composition of the invention (having a concentration of about 3% (w/v)). A similar ratio of volume of the composition to the volume of the brain (about 1/2000- 1/100) can be used for subjects with larger brains, such as human patients. Because the spinal cord is much smaller than the brain, the dosage to the spinal cord would be about 1/10th to 1/100 th of the dosage to the brain.
  • the dose of the biologically active material will vary; suitable amounts for each particular agent will be evident to a skilled worker.
  • kits useful for any of the methods disclosed herein can comprise one or more of the compositions of the invention.
  • the kits comprise instructions for performing the method.
  • Optional elements of a kit of the invention include suitable buffers, pharmaceutically acceptable carriers, or the like, containers, or packaging materials.
  • the reagents of the kit may be in containers in which the reagents are stable, e.g ., in lyophilized form or stabilized liquids.
  • the reagents may also be in single use form, e.g., in single dosage form.
  • the kit comprises a lyophilized block copolypeptide of the invention, and, optionally, one or more suitable molecules in aqueous solution (e.g., a solution comprising a therapeutic agent or an imaging agent, or a submicron particle, such as a vesicle or a double emulsion droplet, which encapsulates an agent of interest).
  • aqueous solution e.g., a solution comprising a therapeutic agent or an imaging agent, or a submicron particle, such as a vesicle or a double emulsion droplet, which encapsulates an agent of interest.
  • the block polypeptide can then be reconstituted with the aqueous solution to form a hydrogel.
  • the kit comprises a lyophilized block copolypeptide of the invention which has been chemically functionalized with a biomolecule of interest and, optionally, an aqueous solution with which the lyophilized block copolypeptide can be reconstituted.
  • DCH diblock copolypeptide hydrogels
  • CNS central nervous system
  • DCH composed of lysine, homoarginine or glutamate in combination with leucine.
  • a range of DCH formulations with rheological properties similar to brain tissue were injected into mouse forebrain and examined after 1 to 8 weeks using light microscopy, immunohistochemistry and electron microscopy, DCH deposits elicited no more gliosis, inflammation, or toxicity to neurons, myelin or axons than did injections of physiological saline.
  • DCH deposits could be varied subtly by altering DCH composition and concentration. For any given DCH formulation, increased concentration correlated with increased gel strength in vitro and increased deposit size in vivo.
  • DCH of lysine and leucine (K m L n ) were selected for detailed analyses because these formed deposits with desirable physical properties and since lysine is routinely used as a substrate for neural cell cultures.
  • Deposits of unmodified K 180 L 20 exhibited time dependent in-growth of blood vessels and of certain glial cells, and limited in-growth of nerve fibers.
  • DCH with different properties can be prepared by varying amino acid compositions, chain lengths, and by conjugation of molecules with specific properties to amino acid side-chain functional groups ( Fig. 1A,B ) (Nowak et al. (2002) ( supra ); Deming (2005) ( supra )).
  • block copolypeptides containing polyelectrolyte segments and hydrophobic helical domains can stabilize formation of hydrated membranes and fibrils that form a robust hydrogel network (Deming (2005) ( supra )).
  • the assembly mechanism elucidated via analysis of structure-property relationships and the use of a range of characterization tools ( Pochan et al.
  • THF Tetrahydrofuran
  • hexanes hexanes
  • methylene chloride were dried by purging with nitrogen and passage through activated alumina columns prior to use ( Pangborn et al. (1996), Safe and convenient procedure for solvent purification, Organometallics 15, 1518-152 ).
  • Co(PMe 3 ) 4 was prepared according to literature procedures ( Klein et al. (1975), Methylcobalt compounds with non-chelating ligands I. Methyltetrakis(trimethylphophine) cobalt and its derivatives. Chemische Berichte 108, 944-955 ).
  • Trimethyliodosilane (TMIS) was purified by distillation and stored over copper shot prior to use.
  • FTIR Fourier Transform Infrared
  • K 180 L 20 (SEQ ID NO:8) to give R 180 L 20 (SEQ ID N0:26).
  • Homoarginine residues can be obtained by converting the ⁇ -amino groups in lysine side chains to guanidinium groups using O -methylisourea ( Kimmel (1967) Guanidination of proteins, Methods Enzymol 11, 584-589 ; Beardsley et al. (2002) Optimization of guanidination procedures for MALDI mass mapping. Anal Chem 74,1884-1890 ).
  • the typical conversion efficiency is 80-85% yield ( Beardsley et al. (2002) Optimization of guanidination procedures for MALDI mass mapping, Anal Chem 74, 1884-1890 ).
  • the guanidinylation reaction mixture was prepared by mixing O -methylisourea solution (3.0 g, 27 mmol of O-methylisourea hydrochloride in 10 mL of DI water, pH adjusted to 10.5) with aqueous K 180 L 20 solution (160 mg, 3.8 ⁇ mol of K 180 L 20 (SEQ ID NO:8) in 20 mL of pyrogen free water) in a sterilized 100-mL round bottom flask. The final pH was adjusted to 10.5 and the reaction was carried out at 37°C under stirring for one week. Guanidinylation was terminated by acidifying the solution with hydrochloric acid.
  • the mixture was then transferred to a dialysis bag (Regenerated Cellulose, Spectra/Por, MWCO 6-8 kDa) that was placed in a sterile 4-liter container of pyrogen free water. The water was replaced twice a day for 5 days. The solution was then freeze dried to yield the product as a spongy solid.
  • the extent of guanidinylation was determined with gas chromatography by quantifying the residual lysines in R 180 L 20 hydrolyzate.
  • the guanidinium groups of homoarginine are not derivatized with ethylchloroformate (ECF), and so these residues do not elute from the column (Husek, 1991).
  • ECF ethylchloroformate
  • K 180 L 20 SEQ ID NO:8
  • Texas Red-K 180 L 20 SEQ ID NO:8
  • Fluorescent tagging of lysine- ⁇ -amine groups was performed using Texas Red sulfonyl chloride in DMF (10 mg/mL).
  • K 180 L 20 (SEQ ID NO:8) powder (130 mg, 3.2 ⁇ mol) was dissolved in aqueous NaHCO 3 (30 mL, 0.1 M).
  • 5 equivalents of Texas Red per chain corresponding to 2.8% of the available lysine amines
  • LiBr (1 g) and HCl 500 ⁇ l, 12 N
  • the sample covered in Al foil to protect from light, was dialyzed (MWCO 6-8 kDa) for 5 days with pyrogen free water changed every 12 hours.
  • the functionalized polymer (Texas Red-K 180 L 20 ) was isolated by lyophilization to give a slightly yellow powder (130 mg).
  • the derivatives were extracted into the organic phase by vortexing and then centrifugation. For each sample, an aliquot of the organic phase (1 ⁇ L) was injected into the gas chromatography capillary column (Phenomenex ZB-AAA 10 m x 0.25 mm ID Amino Acid Analysis column). A Shimadzu GC-17A gas chromatograph with flame ionization detector (FID) and auto sampler was employed. Sample injection mode (1:15) was used for each analysis. The injector and detector temperatures were 300°C and 325°C, respectively. The oven temperature was programmed to increase from 110°C to 320°C stepwise (32°C/min). Helium was used as the carrier gas with a constant flow rate (1.5 mL/min). Standard calibrations for each amino acid were established using individual amino acid standard with six different concentrations ranging from 0.5 mM to 10 mM using identical derivatization and analysis as the copolypeptide hydrolyzates. Each sample or standard was measured in triplicate.
  • mice received terminal anesthesia by barbiturate overdose and were perfused transcardially with phosphate buffered saline (PBS) followed by 10% formalin in PBS. Brains were removed, post-fixed for a further 2 hours and cryoprotected in buffered 30% sucrose overnight. 40 ⁇ m coronal frozen sections were prepared using a cryostat microtome (Leica, Nussloch, Germany) and processed for various kinds of histological evaluation as described previously (Myer et al. (2006) ( supra ).
  • PBS phosphate buffered saline
  • Brightfield immunohistochemistry was performed using biotinylated secondary antibodies (Vector, Burlingame, CA), biotin-avidin-peroxidase complex (Vector) and diaminobenzidine (DAB, Vector) as the developing agent.
  • Primary antibodies were: rabbit anti-polylysine (1:5000, Dr. Katopodis ( Katopodis et al. (2002) Removal of anti-Galalpha1,3Gal xenoantibodies with an injectable polymer, J Clin Invest 110.
  • rabbit anti-GFAP (1:20,000; Dako, Carpinteria, CA), rat anti-GFAP (1:5000, Zymed Laboratories); rat antimouse CD45 (1:2000; PharMingen, La Jolla, CA); rat anti-CD3 (1:2000; PharMingen, La Jolla, CA); mouse anti-NeuN (1:5000 Millipore); rabbit anti-PECAM1 (1:400; PharMingen, La Jolla, CA); rabbit anti-NG2 (1:2500; Millipore). Staining with cresyl violet or luxol fast blue was conducted according to standard procedures.
  • mice were perfused, after terminal barbiturate overdose, with buffered 2% paraformaldehyde and 1.0% glutaraldehyde. Tissue blocks were dissected, post-fixed for 24 hours and processed for electron microscopy as described in detail previously (Havton et al. (2005). Briefly, sections were osmicated in 1% osmium tetroxide, dehydrated and plastic-embedded in Epon. Semithin sections were cut, stained with toluidine blue, and examined in the light microscopy to identify biopolymer injection sites.
  • Tissue blocks were trimmed and ultrathin sections (60-70nm) were serially collected on formvar-coated copper one-hole grids, counterstained with uranyl acetate and lead citrate, and examined in a JEOL 100 CX transmission electron microscope.
  • the CPN contains a large population of neuronal cell bodies intermingled with bundles of myelinated axons that provides a good site to evaluate DCH integration with, and effects on, host CNS tissue, including degree of induction of gliosis and inflammation, potential toxicity towards neurons, myelin or axons and ability to act as a scaffold for growth and migration of host cells and nerve fibers, as discussed below.
  • mice injected with DCH into the CPN exhibited no detectable adverse effects and were behaviorally indistinguishable from mice injected with an equivalent amount of physiological saline during routine monitoring over survival periods from 1 to 8 weeks after injection.
  • DCH DCH of various compositions and concentrations
  • K 200 SEQ ID NO:39
  • K 190 L 10 SEQ ID NO:7
  • K 170 L 30 -0.5% and K 160 L 40 -0.25% which exhibited similar gel strengths in vitro
  • K 160 L 40 -0.5% which exhibited a much higher gel strength in vitro
  • K 170 L 30 -1% which exhibited a greater gel strength in vitro (Table 3)
  • K 180 L 20 was able to self-assemble and form small uneven deposits in vivo.
  • electron microscopy demonstrated the presence within the 3%-K 180 L 20 deposits after 4 or 8 weeks in vivo of cell nuclei with the ultrastructural characteristics ( Peters et al. (1991) The fine structure of the nervous system, Third Edition. New York: Oxford University Press ) of endothelia and different types of glia. Immunohistochemical evaluation of the type of cells migrating into the deposits is described below.
  • DCH trigger minimal gliosis, inflammation or toxicity
  • Injuries or foreign materials in the CNS elicit reactive gliosis and inflammation, characterized by astrocyte reactivity, microglial reactivity and in severe cases by recruitment of blood borne inflammatory cells.
  • DCH formulations we used immunohistochemistry for GFAP as a marker for reactive astrocytes, CD45 as a marker for microglia and blood borne inflammatory cells and CD3 as a marker for T cells.
  • GFAP a marker for reactive astrocytes
  • CD45 as a marker for microglia and blood borne inflammatory cells
  • CD3 as a marker for T cells.
  • the penetrating injury of inserting cannulae and injecting 2 ⁇ l of sterile physiological saline into the CPN consistently induced a moderately pronounced response of astrocyte reactivity with up-regulation of GFAP expression and hypertrophy of astrocytes when compared with uninjected tissue.
  • images were taken of tissue sections through the CPN immunohistochemically stained for the reactive astrocyte marker, GFAP.
  • This astrocyte reactivity was maximal at 1 week, extended for several hundred micrometers into host tissue adjacent to the cannula insertion sites, and had resolved considerably by 8 weeks. In all cases, the reactive astrogliosis was restricted to the immediate vicinity of the injection cannula and never extended beyond the borders of the CPN.
  • CD45 positive leukocytes To evaluate more specifically the nature of the globoid CD45 positive leukocytes observed in the vicinity of injections of either saline or DCH, we used immunohistochemistry for CD3 to detect T cells and to assess immune surveillance. In this study, images were taken of tissue sections through the CPN immunohistochemically stained for the T-cell marker, CD3. As expected, we found a small number of CD3 positive leukocytes in the immediate vicinity of the saline injections sites at all time points and that T cells continued to monitor the sites of cannula insertion and saline injection even after 8 weeks).
  • DCH formulations including 1%- K 180 L 20 , 5%-K 180 L 20 , 0.5%-K 170 L 30 , 1%-K 170 L 30 , 0.25%-K 160 L 40 , 0.5%-K 160 L 40 , 3%-R 180 L 20 , 5%-R 180 L 20 , 1%-E 180 L 20 and 2%-E 180 L 2 , which all elicited no greater levels of toxicity to neurons, myelin or axons than those observed after injections of physiological saline.
  • 3%-K 180 L 20 deposits were packed with cells. Both immunohistochemical staining for PECAM and electron microscopy indicated that 3%-K 180 L 20 deposits were well vascularized by 4 weeks and remained so after 8 weeks, and contained many well-formed blood vessels with normally appearing endothelial cells throughout the deposits. After 4 and 8 weeks, there were many NG2-positive cells scattered within the 3%-K 180 L 20 deposits. After 4 and 8 weeks, many CD45 positive cells were evenly distributed throughout the 3%-K 180 L 20 deposits and most of these had the appearance of stellate-shaped microglia that extended processes along blood vessels or among other cells.
  • astrocytes the other main glial cell type with migratory capacity, only migrated in small numbers into 3%-K 180 L 20 deposits, even after 8 weeks in vivo.
  • NFM neurofilament M
  • TH tyrosine hydroxlase
  • NFM or TH fibers in the CPN in immediate contact with DCH deposits was indistinguishable from that surrounding injection sites of saline.
  • both NFM and TH fibers were found in small numbers to grow into DCH deposits with time.
  • deposits of unmodified 3%-K 180 L 20 exhibited no appreciable in-growth of nerve fibers positive for either NFM or TH.
  • small numbers of single NFM-positive fibers were found crossing from the host into the deposits along the borders, and occasional NFM fibers were found within the center of deposits, generally in association with blood vessel walls.
  • DCH are highly versatile and can easily be synthesized and dissolved in a wide variety of formulations that have rheological properties in the range of CNS tissue.
  • DCH can be injected as liquids through small-bore cannulae and rapidly re-assemble into well-formed deposits in vivo, allowing facile and minimally invasive in vivo delivery.
  • size and physical properties (density/porosity) of DCH deposits in vivo can be varied subtly and predictably by small alterations in DCH composition or concentration.
  • both the gelation strength in vitro, and the size and porosity of deposits in vivo could be varied in a predictable manner by altering either the (i) amino acids used in the hydrophilic copolymer chains, (ii) ratio of charged to hydrophobic amino acid residues, or (iii) concentration of DCH formulations.
  • K m L n DCH for detailed analyses because polylysine is routinely used as a substrate for neural cell cultures (Yavin et al. (1974) ( supra ); Svendsen et al. (1994) ( supra ); Sanjana et al. (2004) ( supra )) and is a good candidate to interact well with neural tissue in vivo.
  • DCH formulation of K 180 L 20 as exhibiting good deposit formation with desirable properties that could be varied subtly according to weight percent concentration to achieve different degrees of deposit consistency and porosity that may be suitable for different applications.
  • DCH are biocompatible, nontoxic and degrade over time in vivo
  • DCH are composed of chains of naturally occurring amino acids linked by conventional peptide bonds and would be expected to degrade over time in vivo.
  • Our electron microscopic findings confirmed this expectation and showed that (i) after 1 week in vivo, deposits of 3%-K 180 L 20 consisted entirely of dense networks extracellular DCH fibrils, (ii) the density of extracellular fibrils decreased after 2 and 4 weeks as cellular in-growth increased, and (iii) by 8 weeks there were no detectable extracellular DCH fibrils, but there were many cells with clear vacuoles, suggesting that the DCH had either dissolved away or been phagocytosed.
  • DCH have potential for use as scaffolds to promote cell and axon growth in the CNS. This capacity may be enhanced by functionalizing the DCH through attachment of specific molecular epitopes that are able to provide matrix support with specific permissive or inhibitory migration guidance cues, of which many have been identified and some tested in combination with biomaterials in the CNS ( Schmidt et al. (2003) Neural tissue engineering: strategies for repair and regeneration, Annu Rev Biomed Eng 5, 293-347 ; Zhang et al. (2005) Tissue-engineering approaches for axonal guidance, Brain Res Brain Res Rev 49, 48-64, 2005 ; Zhong et al.
  • DCH can be used for CNS applications
  • a number of structural and functional features are likely to be required of materials for CNS applications including: non-toxicity; biocompatability; degradability; injectability; tunable mechanical properties (e.g. rigidity and porosity); ability to interact with cells; tunable sustained release of diffusible biologically active agents; and tunable presentation of extracellular matrix cues to support and guide cell migration or nerve fiber growth.
  • Materials currently investigated for CNS applications have shown promise but also limitations. Materials prepared from natural polymers, e.g. hyaluronic acid (HA) ( Hou et al.
  • HAMC hyaluronan/methylcellulose
  • DCH compare favorably with these other materials and exhibit many characteristics desirable for CNS applications. DCH shear thin to liquids during injection and rapidly re-assemble into well-formed deposits that persist in vivo for prolonged periods of time. DCH are biocompatible, and integrate well with host cells and tissue. The time-dependent in-growth of certain cell types into DCH deposits suggests the potential for DCH deposits to serve as scaffolds for cell migration in the CNS in a cell or nerve fiber specific manner if they are functionalized with the appropriate migration guidance cues. DCH scaffolds may also have the potential to support grafted cells.
  • the amino acid side-chains of DCH provide the potential for functionalization of DCH with a vast array of bioactive molecules, thus providing DCH with the potential for wide biological versatility, for example to serve as depots for sustained molecular release or to present covalently bound matrix molecules to support and guide cell migration.
  • DCH can readily be labeled with probes to mark their location in vivo, as shown here with the fluorescent probe Texas Red, and which could be done using markers available for in vivo imaging.
  • the synthesis of DCH can be modified readily and predictably to achieve subtly different mechanical properties, while retaining basic attributes. This important feature enables the in vivo properties of DCH to be fine-tuned in a context specific manner as might be required by different applications in the CNS.
  • different mechanical properties may be required for providing depot delivery with sustained release of diffusible molecules, as compared with providing a scaffold to bridge cell and nerve fiber migration across scar tissue or to fill wound cavities.
  • attributes of sustained release and scaffold may be required together.
  • Fine-tuning of mechanical properties may also be important to achieve optimal integration of materials with host CNS tissues that have subtly different properties, for example after different insults such as traumatic or ischemic injury, or degenerative disease. Mechanical properties of extracellular surroundings can markedly, and differentially, influence growth properties of specific neural and glial cell types (Discher et al. (2005) ( supra ); Georges et al. (2006) ( supra )).
  • Example I DCHs exhibit little or no detectable in vivo toxicity and integrate well with host cells and nerve fibers in the forebrain. These attributes, together with the functionality of DCH, amino acid side chains, which allow conjugation of molecules to DCH indicate that DCHs are good candidates for development of therapeutic depots in the CNS.
  • the incorporated therapeutics e.g. nucleic acids, oligopeptides, proteins, enzymes, carbohydrates, viruses, cells, etc.
  • this release occurs over an extended time to reduce the need for additional administrations of the therapeutics.
  • chondroitinase ABC chondroitin sulfate proteoglycans
  • CSPGs chondroitin sulfate proteoglycans
  • proteins or peptides can also be covalently attached to polymers through a cleavable linkage and then released afterwards when the linkage is cleaved.
  • This covalent attachment can be achieved by reacting the different side chain functionalities of polymers with the amino acids of the proteins or peptides (e.g. aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, tyrosine, methionine, and tryptophan).
  • any derivatization should result in a protein that performs exactly as it would in its unmodified form, but with the added functionality imparted by whatever is conjugated to it ( Hermanson, Bioconjugate Techniques, Academic Press, Inc., 2008 ).
  • the most common functional groups present in cross-linking or coupling reagents are groups that are able to react with amine-containing molecules.
  • An amine-coupling process can be used to conjugate with nearly all protein or peptide molecules as well as a host of other macromolecules. Most of these reactions are rapid and occur in high yield to give stable amide or secondary amine bonds (Hermanson (2008) ( supra )).
  • Carboxylate groups activated as N -hydroxysuccinimide (NHS) esters are highly reactive toward amine nucleophiles. NHS ester-containing molecules that react with primary or secondary amines release the NHS leaving group and form stable amide and imide linkages.
  • a carboxylate-containing molecule can be transformed into an active ester functional group by reaction in the presence of NHS ( Figure5 ).
  • a peptide or protein can thus be conjugated through its carboxylate or amine groups to complimentary groups on either lysine or glutamate based DCHs using amine coupling.
  • the photochemically/thermally-induced version of this reaction is known to proceed by a radical mechanism to give an anti-Markovnikov-type thioether.
  • the click status of this reaction is supported by it being highly efficient and orthogonal to a wide range of functional groups, as well as for being compatible with water and oxygen.
  • Conducting the thiol-ene reactions under benign reaction conditions and without the use of any metal catalysts allows for an environmentally friendly process to be developed, further enhancing the attractive nature of this process.
  • Alkene groups can be linked to the functional groups of hydrophilic side chains in DCHs via different approaches. These include a permanent or cleavable linkage to the NCA monomer or to the DCH polymer.
  • cleavable linkage that would respond to different stimuli such as pH, temperature, or other molecules.
  • a cleavable crosslinker 4-allyloxy-4-oxo-butanoic acid for the initial trial. It has an alkene group on one end that can be used for thiol-ene coupling to thiolated proteins. Its other end is a carboxylic group that can be coupled to the N a- amine on the side chain of lysine in DCH. There is an ester group in the middle of the linker that should hydrolyze slowly over time under physiological conditions.
  • 3-butenoic acid can be conjugated to the N ⁇ -amine in N ⁇ -Z-lysine via amine coupling and then cyclized to form the NCA monomer with (1-chloro-2-dimethylamino-2-methyl) propene.
  • the alkene-functionalized NCA monomer can then be copolymerized using Co(PMe 3 ) 4 to obtain alkene-functionalized DCH polymers.
  • 3-butenoic acid can be attached to N ⁇ -amine in the poly-L-lysine segments of DCH via amine coupling.
  • Subunits may be held together by noncovalent contacts, such as hydrophobic or ionic interactions, or by covalent disulfide bonds formed by a cysteine residue of one polypeptide chain being cross-linked to a cysteine sulfhydryl of another chain.
  • noncovalent contacts such as hydrophobic or ionic interactions
  • covalent disulfide bonds formed by a cysteine residue of one polypeptide chain being cross-linked to a cysteine sulfhydryl of another chain.
  • chemical modification of amino acid constituents of a protein may also cause significant disruption of its overall 3D structure. It is then often desirable to introduce additional functional groups onto proteins for conjugation.
  • Traut's reagent 2-iminothiolane
  • 2-iminothiolane is a cyclic imidothioester that can react with primary amines in a ring-opening reaction that generates a free sulfhydryl without breaking the native disulfide linkages inside a protein.
  • the newly generated free sulfhydryl can be conjugated to the alkene groups on the side chains of DCHs via the thiol-ene reaction.
  • Thiol-ene coupling is a sulfur radical addition to an alkene group to form a thioether bond.
  • the reaction is easily carried out in an aqueous environment at physiological pH and low temperature (about 4°C to room temperature) in a reasonable reaction time period.
  • thiol-ene coupling has been known for many years, reaction conditions suitable for the bioconjugation of proteins had not been well established prior to the present application.
  • most reported TEC reactions were carried out in organic solvents with strong UV light at high temperature in time periods of days and even months (Gress et al , (2007) ( supra ); Killops et al. (2008) ( supra ); Campos et al.
  • K 180 L 20 powder was dissolved in physiological saline at the concentration of 6 %.
  • ChABC was reconstituted in physiological saline at a concentration of 100 U/mL.
  • 50 ⁇ L of chABC solution was added to 50 ⁇ L of K 180 L 20 gel under gently stirring.
  • the final gel contains 3%- K 180 L 20 and 50 U/mL of chABC.
  • a chABC and K 180 L 20 mixture was prepared as described above. 10 equiv. of NHS and 10 equiv. of EDC were then added to the mixture with stirring for 24 hours. The mixture was used directly.
  • NHS ester of 3-butenoic acid was synthesized based following literature procedure. 32 3-butenoic acid (1.0 g, 1.2 mmol) and NHS (1.47 g, 1.28 mmol) were dissolved in THF (30 ml). The solution was cooled in an ice bath and N,N'-dicyclohyxylcarbodiimide (DCC, 2.4 g, 1.2 mmol) was added under stirring. A white precipitate, dicyclohexylurea (DCU), formed after ⁇ 5 min and the mixture was stirred for 1 hour at 0 °C. The reaction proceeded at 4 °C overnight without any agitation. DCU was then removed by filtration.
  • DCC N,N'-dicyclohyxylcarbodiimide
  • ⁇ -Z-L-lysine 2.5 g, 9.0 mmol
  • NaHCO 3 0.8 g, 9.9 mmol
  • the alkene NHS-ester (1.9 g, 6.8 mmol) prepared as above was dissolved in THF and then added to the mixture containing ⁇ -Z-L-lysine. The reaction proceeded at room temperature overnight. THF was then removed under vaccum. HCl (1 N) was added to adjust its pH to ⁇ 3. 50 mL of ethylacetate was added to the solution and transferred to a 250-mL separatory funnel. The organic and aqueous phases were separated and individually collected.
  • the product was in the organic phase containing ethylacetate. Fresh ethylacetate was added to the aqueous phase and the separation and collection procedure was repeated for another few times. All of the ethylacetate solutions containing the product were combined together and filtered through a glass filter. The filtrate was then collected. Most of the ethylacetate was removed by rotorvap and then extensively dried under vacuum. We tried to re-crystallize the product by using the solvent pair of ethylacetate/hexane (10 mL/50 mL) at -20 °C for one month. Some white powder appeared over time. The solvent was removed under vacuum and dry white powder was obtained (2.9 g).
  • chABC Seikagaku Corp., East Falmouth, MA, USA
  • control bacteria enzyme penicillinase, Sigma, St. Louis, MO, USA
  • Primary antibodies were: monoclonal mouse anti-2B6 (1:3,000; Seikagaku); Bio-WFA (1:2,500; Sigma-Aldrich).
  • Bioconjugation work was performed with horseradish peroxidase (HRP), ovalbumin, and laminin-1. These proteins were conjugated (at the ratio ranging from 1 to 0.01 proteins per polymer chain) to K 180 L 20 via amine coupling. The polymers were then freeze-dried after dialysis. Upon rehydration, some loss in protein activity was observed. Since freeze-drying (lyophilization) removes water from a frozen sample by sublimation and desorption, this could cause structural changes in the conjugated proteins and the gel network. Routine optimization of freeze drying of proteins conjugated to DCH will be carried out in order to improve the ability to store these sample as lyophilized powders.
  • HRP horseradish peroxidase
  • ovalbumin ovalbumin
  • laminin-1 laminin-1
  • ChABC belongs to a family of bacterial enzymes which cleave glycosaminoglycan (GAG) chains by a ⁇ -elimination mechanism.
  • This enzyme digests chondroitin sulfate (CS)-A, -B and -C isoforms which correspond to chondroitin 4-sulfate (C4S), dermatan sulfate (DS) and chondroitin 6-sulfate (C6S) respectively ( Yamagata et al. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases, J Biol Chem 243, 1523-1535 ).
  • the intact CSPGs could be recognized by the N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA) antibody, and the "stubs" on C4S core proteins remaining after digestion of CSPGs by chABC sould be recognized by the 2B6 antibody.
  • WFA N-acetylgalactosamine-binding Wisteria floribunda agglutinin
  • Example III Biocompatibility of amphiphilic diblock copolypeptide hydrogels in the central nervous system/ use as scaffolds
  • Angiogenesis refers to the formation of new capillary blood vessels by a process of sprouting from pre-existing vessels or precursor cells of endothelia that participate in embryogenesis as well as normal and pathological vessel formation in postnatal life.
  • the process of angiogenesis is orchestrated by endothelial cells and neighboring mural cell types via various growth factors and extracellular matrix proteins.
  • endothelial cells and neighboring mural cell types via various growth factors and extracellular matrix proteins.
  • the ile-lys-val-ala-val (IKVAV)-containing site on the long arm of the laminin ⁇ 1 chain ( Tashiro et al. (1989) A synthetic peptide containing the (SEQ ID NO:1) IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth, J Biol Chem 264, 16174-16182 ) has also been reported to promote cell adhesion, neurite outgrowth, and angiogenesis (Tashiro et al. (1989) ( supra ); Sephel et al. (1989) Laminin A chain synthetic peptide which supports neurite outgrowth, Biochem Biophys Res Common 162. 821-829 ; Grant et al.
  • Laminin-1 promotes angiogenesis in synergy with fibroblast growth factor by distinct regulation of the gene and protein expression profile in endothelial cells, J Biol Chem 279, 23766-23772 ).
  • Laminin-1 and IKVAV were each incorporated into DCHs to enhance functionality and test the feasibility of DCHs as scaffolds in the CNS.
  • Laminin-1 (R&D Systems, Minneapolis, MN, USA) or (SEQ ID NO:1) IKVAV (American Peptide, Sunnyvale, CA, USA) was conjugated to K 180 L 20 via amide bond coupling.
  • the final concentration of laminin was 0.5 mg/mL.
  • the conjugation ratio of (SEQ ID NO:1) IKVAV was 5 molecules per K 180 L 20 polymer chain.
  • samples of unmodified or IKVAV (SEQ ID NO:1) -conjugated K 180 L 20 were injected into both sides of the forebrain.
  • Laminin decorated 3%-K 180 L 20 were injected into one side of the forebrain only.
  • Laminin-1 decorated 3%-K 180 L 20 significantly promoted cell in-migration and angiogenesis in the forebrain at 1 week after injection. These results are comparable to the previously demonstrated in-migration of cells and angiogenesis of unmodified 3%-K 180 L 20 after 4 weeks in vivo.
  • Example IV Loading of DCH with bioactive proteins a release of these cargos in vivo .
  • Providing sustained local, site-restricted release of diffusible bioactive molecules, such as polypeptide growth factors, function blocking hybrid proteins, oligonucleotides and certain enzymes are applications well-suited to injectable biomaterial depots such as DCH.
  • This study examined (a) the release kinetics of bioactive molecules from DCH depots in vitro and (b) the efficacy of molecular delivery from DCH depots in vivo .
  • the uptake of polypeptide nanocarriers plus oligonucleotides into CNS cells in vivo was evaluated.
  • DCH depots will release molecules into the surrounding host CNS tissue and that the release rate can be varied and controlled in various ways, either by using cleavable linkers to bind molecules to the DCH or by loading molecules into vesicles or emulsions. Different kinds of molecules require different delivery methods, and optimizing delivery can be accomplished with a combination of routine, conventional informed design and empirical testing.
  • This Example demonstrates sustained extracellular delivery release of different proteins such as enzymes, function blocking hybrid molecules and growth factors. Delivery of proteins was evaluated by comparing release from DCH depots under three different conditions: (i) dissolved in DCH; (ii) bound to DCH by permanent linker; and (iii) bound to the DCH by a reversible linker. Release kinetics associated with these three conditions was evaluated in vitro by measuring the escape of fluorescence-tagged proteins (Texas Red labeled BSA) from DCH across a border of dialysis tubing porous to the proteins but not to DCH. Dialysis tubing porous to the tags but not the proteins was used as control. Release in vivo was characterized after injection of different DCH formulations into the mouse forebrain as done previously. Different DCH formulations were injected into the forebrain and release of bioactive molecules in vivo was determined by analysis of bioactivity or direct protein assay by ELISA.
  • fluorescence-tagged proteins Texas Red labeled BSA
  • Chondroitinase is an enzyme that degrades the axon growth inhibitors, chondroitin sulfate proteoglycans (CSPG). Release of bioactive chondroitinase from DCH depots in vivo is demonstrated in Figure 15 .
  • DCH depots can effectively deliver bioactive proteins dissolved in the DCH; and bioactive molecules gradually escape from DCH into surrounding host tissue and exert effects with full potency.
  • Example V Loading of DCH with nanocarriers (vesicles and emulsions) to release or promote cellular uptake of encapsulated or entrapped cargos in vivo .
  • bioactive molecules with therapeutic potential for the CNS require encapsulation to prevent their degradation and need to be delivered intracellularly (e.g . siRNA and plasmids).
  • siRNA and plasmids e.g. siRNA and plasmids.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Psychology (AREA)
  • Dispersion Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Optics & Photonics (AREA)
  • Radiology & Medical Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)

Claims (28)

  1. Composition convenant à l'administration au système nerveux central (SNC), comprenant un hydrogel de copolypeptide séquencé, dans laquelle le copolypeptide séquencé comprend entre 100 et 600 acides aminés,
    qui comprend une matière biologiquement active qui est mélangée avec l'hydrogel et/ou qui est attachée au squelette polypeptidique de l'hydrogel,
    dans laquelle le copolypeptide séquencé comprend au moins deux segments polypeptidiques distincts choisis parmi des fractions hydrophiles et des fractions hydrophobes liées de manière covalente, dans laquelle la quantité de copolymère varie de 0,1 à 5 % poids/volume, et dans laquelle la composition convient à une administration au SNC.
  2. Composition selon la revendication 1, dans laquelle le copolypeptide séquencé est un copolypeptide diséquencé.
  3. Composition selon la revendication 1, dans laquelle la matière biologiquement active est un agent thérapeutique, un agent de diagnostic, ou une protéine ou un peptide qui crée un repère de guidage de migration.
  4. Composition selon la revendication 1, dans laquelle la matière biologiquement active est hydrophile et est mélangée à l'hydrogel.
  5. Composition selon la revendication 1, dans laquelle la matière biologiquement active est hydrophobe et est mélangée à l'hydrogel.
  6. Composition selon la revendication 1, dans laquelle la matière biologiquement active est piégée dans un nanosupport, qui est mélangé à l'hydrogel.
  7. Composition selon la revendication 6, dans laquelle le nanosupport est une vésicule ou une gouttelette double émulsion.
  8. Composition selon la revendication 1, dans laquelle la matière biologiquement active est attachée de manière covalente au squelette polypeptidique de l'hydrogel.
  9. Composition selon la revendication 8, dans laquelle la matière biologiquement active est un peptide, un polypeptide, un sucre, un oligosaccharide, un polysaccharide, une glycoprotéine, un oligonucléotide, un agent de contraste d'IRM, un radioisotope ou une sonde fluorescente.
  10. Composition selon la revendication 8, dans laquelle la matière biologiquement active est un peptide ou un polypeptide qui fonctionne comme un repère de guidage de migration.
  11. Composition selon la revendication 10, dans laquelle le polypeptide ou peptide qui fonctionne comme un repère de guidage de migration est une laminine, ou un peptide bioactif de celle-ci, et le DCH attaché au peptide ou au polypeptide forme un échafaudage qui supporte la migration cellulaire, la croissance de fibres nerveuses et/ou la formation de vaisseaux sanguins.
  12. Composition selon la revendication 1, dans laquelle la matière biologiquement active est un agent de diagnostic ou un agent thérapeutique, et est mélangée dans l'hydrogel, ou est attachée de manière covalente au squelette polypeptidique de l'hydrogel.
  13. Composition selon la revendication 12, dans laquelle l'agent thérapeutique est la chondroïtinase ABC (chABC).
  14. Composition selon la revendication 13, dans laquelle la chABC est attachée de manière covalente au squelette polypeptidique, et l'attache covalente est réalisée par un couplage thiol-ène (TEC) d'un groupe thiol de chABC qui a été thiolé à un groupe fonctionnalisé alcène du squelette polypeptidique.
  15. Composition selon l'une quelconque des revendications 1 à 14, dans laquelle le copolypeptide séquencé comprend la poly-L-leucine (L), la poly-L-isoleucine (I), la poly-L-phénylalanine (F), la poly-L-alanine (A), ou la poly-L-valine (V), ou un mélange de ces acides aminés, en tant que domaine hydrophobe, et la poly-L-lysine (K), la poly-L-ornithine (0), la poly-L-arginine (R), la poly-L-homoarginine (RH) ou le poly-L-glutamate (E), ou un mélange de ces acides aminés, en tant que domaine hydrophile.
  16. Composition selon l'une quelconque des revendications 1 à 14, dans laquelle le copolypeptide séquencé comprend la poly-D-leucine (L), la poly-D-isoleucine (I), la poly-D-phénylalanine (F), la poly-D-alanine (A), ou la poly-D-valine (V), ou un mélange de ces acides aminés, en tant que domaine hydrophobe, et la poly-D-lysine (K), la poly-D-ornithine (0), la poly-D-arginine (R), la poly-D-homoarginine (RH) ou le poly-D-glutamate (E), ou un mélange de ces acides aminés, en tant que domaine hydrophile.
  17. Composition selon l'une quelconque des revendications 1 à 14, dans laquelle le copolypeptide séquencé comprend de la poly-L-leucine (L) en tant que domaine hydrophobe, et de la poly-D/L-lysine racémique (rac-K) en tant que domaine hydrophile.
  18. Composition selon l'une quelconque des revendications 1 à 14, dans laquelle le copolypeptide séquencé consiste essentiellement en de la poly-L-leucine (L) en tant que domaine hydrophobe et de la poly-L-lysine (K) en tant que domaine hydrophile.
  19. Composition selon l'une quelconque des revendications 1 à 14, dans laquelle le copolypeptide séquencé consiste essentiellement en K180L20 (SEQ ID N° : 8).
  20. Procédé de préparation d'une composition de la revendication 1, comprenant l'attache covalente d'un peptide ou d'une protéine d'intérêt au squelette polypeptidique, par
    a) thiolation du peptide ou de la protéine d'intérêt,
    fonctionnalisation alcène d'un groupe du squelette polypeptidique, et
    couplage thiol-ène (TEC) d'un groupe thiolé du peptide ou de la protéine qui a été thiolée en un groupe fonctionnalisé alcène du squelette polypeptidique, ou
    b) thiolation d'un groupe du squelette polypeptidique,
    fonctionnalisation alcène du peptide ou de la protéine d'intérêt, et
    couplage thiol-ène (TEC) d'un groupe thiolé du squelette polypeptidique qui a été thiolé en un groupe fonctionnalisé alcène du peptide ou de la protéine.
  21. Procédé selon la revendication 20, dans lequel le TEC est réalisé dans une solution aqueuse à pH physiologique et à environ 4 °C à température ambiante.
  22. Procédé de préparation d'une composition de la revendication 1, comprenant l'attache covalente d'un peptide ou d'une protéine d'intérêt sur le squelette polypeptidique par couplage d'amine.
  23. Composition selon la revendication 13 ou la revendication 14, à utiliser dans l'introduction de chABC biologiquement active dans un cerveau in vivo.
  24. Procédé de création d'un échafaudage pour une migration cellulaire dans le système nerveux central (SNC), comprenant la fonctionnalisation d'un hydrogel copolypeptidique séquencé de la revendication 1 avec une protéine ou un peptide qui crée un repère de guidage de migration.
  25. Procédé selon la revendication 24, dans lequel la protéine ou le peptide qui crée un repère de guidage de migration est une laminine, ou un peptide biologiquement actif de celle-ci.
  26. Echafaudage selon la revendication 10 ou la revendication 11, à utiliser dans la stimulation de la migration cellulaire ou de l'angiogenèse, ou à utiliser dans la stimulation de la croissance interne de fibres nerveuses, dans un cerveau.
  27. Echafaudage à utiliser selon la revendication 26, qui renforce la plasticité axonale et/ou améliore la récupération fonctionnelle après réparation de nerfs périphériques à l'issue d'une lésion de la moelle épinière.
  28. Kit, comprenant
    (a) un copolypeptide séquencé de la revendication 1, qui a été lyophilisé, et
    une solution aqueuse comprenant un agent thérapeutique, un agent d'imagerie, ou une particule submicronique encapsulant un agent d'intérêt, avec lequel le copolypeptide séquencé lyophilisé peut être reconstitué ; ou
    (b) un copolypeptide séquencé de la revendication 1, qui a été chimiquement fonctionnalisé avec une biomolécule d'intérêt, et qui a été lyophilisé, et
    une solution aqueuse avec laquelle le copolypeptide séquencé lyophilisé fonctionnalisé peut être reconstitué.
EP10744302.0A 2009-02-18 2010-02-18 Hydrogels de co-polypeptide à double bloc synthétique pour une utilisation dans le système nerveux central Not-in-force EP2400974B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US15352709P 2009-02-18 2009-02-18
US18667109P 2009-06-12 2009-06-12
PCT/US2010/024603 WO2010096572A2 (fr) 2009-02-18 2010-02-18 Hydrogels de co-polypeptide à double bloc synthétique pour une utilisation dans le système nerveux central

Publications (3)

Publication Number Publication Date
EP2400974A2 EP2400974A2 (fr) 2012-01-04
EP2400974A4 EP2400974A4 (fr) 2012-12-12
EP2400974B1 true EP2400974B1 (fr) 2017-09-06

Family

ID=42634442

Family Applications (1)

Application Number Title Priority Date Filing Date
EP10744302.0A Not-in-force EP2400974B1 (fr) 2009-02-18 2010-02-18 Hydrogels de co-polypeptide à double bloc synthétique pour une utilisation dans le système nerveux central

Country Status (4)

Country Link
US (2) US8691204B2 (fr)
EP (1) EP2400974B1 (fr)
CA (1) CA2752902A1 (fr)
WO (1) WO2010096572A2 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112013004436A2 (pt) 2010-08-23 2016-06-21 Amicrobe Inc composições e usos de materiais com atividade microbicida alta e toxicidade baixa
US9364995B2 (en) 2013-03-15 2016-06-14 Matterrise, Inc. Three-dimensional printing and scanning system and method
US9945865B2 (en) 2013-03-22 2018-04-17 University Of Florida Research Foundation, Inc. Polymer nanocomposites for early diagnosis of diseases
WO2016054432A1 (fr) * 2014-10-01 2016-04-07 The Regents Of The University Of California Hydrogels de copolypeptides double blocs, non-ioniques et thermosensibles, pour l'administration de molécules et de cellules
ES2974289T3 (es) * 2017-09-28 2024-06-26 Univ California Hidrogeles de polipéptidos de complejo poliiónico y utilizaciones de los mismos
US20210023266A1 (en) * 2018-04-09 2021-01-28 Qrons Inc. Techniques for promoting neuronal recovery
US20220162274A1 (en) * 2019-02-11 2022-05-26 Qualus Research Spa Production and uses of artifical histone h1 for analyzing, diagnosing, treating, and/or preventing senescence
EP3946459A4 (fr) * 2019-03-27 2023-08-16 The Regents of the University of California Mélanges d'hydrogels copolypeptidiques synthétiques
EP3804750A1 (fr) * 2019-10-10 2021-04-14 Universität Heidelberg Composés conjugués pour la prévention et/ou le traitement des infections par le vhb et/ou le vhd, des maladies du foie et pour le ciblage des ntcp
US12090206B2 (en) 2020-04-23 2024-09-17 The Regents Of The University Of California Compositions comprising tri- and penta-block synthetic copolypeptide hydrogels
CN116271187B (zh) * 2023-03-21 2025-07-04 湖北科技学院 双模态显影温敏Pickering乳液介入栓塞材料及其制备方法和应用

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6686446B2 (en) 1998-03-19 2004-02-03 The Regents Of The University Of California Methods and compositions for controlled polypeptide synthesis
WO2001094379A2 (fr) 2000-06-08 2001-12-13 The Regents Of The University Of California Procedes et compositions pour la synthese regulee de polypeptides
EP1407261A4 (fr) * 2001-06-13 2006-11-22 Massachusetts Inst Technology Bioreacteurs pour reactions in vivo
WO2003054508A2 (fr) 2001-10-23 2003-07-03 Anteon Corporation Procede integre, appareil et systemes de generation d'oxygene et d'absorption de dioxyde de carbone
US20030147958A1 (en) 2002-01-29 2003-08-07 Cheol-Hee Ahn Biodegradable multi-block copolymers of poly(amino acid)s and poly(ethylene glycol) for the delivery of bioactive agents
US7204997B2 (en) * 2002-01-29 2007-04-17 Supratek Pharma Inc. Responsive microgel and methods related thereto
CA2601823C (fr) 2005-04-15 2016-01-12 University Of Arizona Peptides therapeutiques pour le traitement de cancers metastatiques
KR101475447B1 (ko) * 2006-12-01 2014-12-23 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 자기-조립 블록 코폴리머의 베시클 그리고 그것의 제조 및 사용 방법
US20080299177A1 (en) * 2007-06-06 2008-12-04 Biovaluation & Analysis, Inc. Supramolecular Complexes for Use in Acoustically Mediated Intracellular Drug Delivery in vivo

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
EP2400974A4 (fr) 2012-12-12
US8691204B2 (en) 2014-04-08
WO2010096572A3 (fr) 2010-11-18
CA2752902A1 (fr) 2010-08-26
EP2400974A2 (fr) 2012-01-04
US20140286865A1 (en) 2014-09-25
US20120093722A1 (en) 2012-04-19
WO2010096572A2 (fr) 2010-08-26

Similar Documents

Publication Publication Date Title
EP2400974B1 (fr) Hydrogels de co-polypeptide à double bloc synthétique pour une utilisation dans le système nerveux central
Yang et al. Biocompatibility of amphiphilic diblock copolypeptide hydrogels in the central nervous system
Guo et al. pH-Responsive charge switchable PEGylated ε-poly-l-lysine polymeric nanoparticles-assisted combination therapy for improving breast cancer treatment
Ghanaati et al. Dynamic in vivo biocompatibility of angiogenic peptide amphiphile nanofibers
DE69425577T2 (de) Multifunktionelle organische polymere
Chung et al. Heparin immobilized porous PLGA microspheres for angiogenic growth factor delivery
Song et al. Sustained local delivery of bioactive nerve growth factor in the central nervous system via tunable diblock copolypeptide hydrogel depots
Kempe et al. In situ forming implants—an attractive formulation principle for parenteral depot formulations
JPH0834747A (ja) コラーゲンベースの注入可能な薬剤送達製剤およびその使用
JP2010519183A (ja) 生理溶液の溶出のためのタンパク質の沈殿を用いる重合
US10688061B2 (en) Formation of delivery agents targeted to degraded elastic fibers
US20200282075A1 (en) Albumin-modified nanoparticles carrying a targeting ligand
Wang et al. Multi-targeted nanogel drug delivery system alleviates neuroinflammation and promotes spinal cord injury repair
Zheng et al. Wound microenvironment-responsive dually cross-linked nanofibrillar peptide hydrogels for efficient hemostatic control and multi-faceted wound management
JP2025513507A (ja) 組織伝導性足場材料
US20180236074A1 (en) Thermoresponsive bioconjugates and their controlled delivery of cargo
TW201725034A (zh) 凝膠組成物及凝膠組成物的製造方法
EP2547690A2 (fr) Peptides d'affinité envers la bmp-2
EP3740525B1 (fr) Particules d'hydrogel à affinité poreuse permettant de réduire la biodisponibilité de molécules biologiques sélectionnées
JP7791819B2 (ja) 生体適合性材料
Boehnke Degradable Hydrogels and Nanogels for the Delivery of Cells and Therapeutics
CN120531857A (zh) 自组装肽组合物及其应用
US11298424B2 (en) Non-ionic and thermoresponsive diblock copolypeptide hydrogels for delivery of molecules and cells
KR102303328B1 (ko) 포켓-형 마이크로 전달체의 제조방법, 이를 이용한 분화방법, 및 이를 포함하는 약학 조성물
Steiert Dynamic Protein-based Nanoparticles for Drug Delivery Applications

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20110913

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20121109

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 31/7004 20060101ALI20121105BHEP

Ipc: A61K 38/00 20060101ALI20121105BHEP

Ipc: A61L 27/22 20060101ALI20121105BHEP

Ipc: A61K 9/113 20060101ALN20121105BHEP

Ipc: A61K 9/06 20060101AFI20121105BHEP

Ipc: A61K 31/7088 20060101ALI20121105BHEP

Ipc: A61L 27/52 20060101ALI20121105BHEP

Ipc: A61K 9/127 20060101ALN20121105BHEP

Ipc: A61L 27/54 20060101ALI20121105BHEP

Ipc: A61K 47/34 20060101ALI20121105BHEP

Ipc: A61K 31/715 20060101ALI20121105BHEP

Ipc: A61K 47/48 20060101ALI20121105BHEP

Ipc: A61K 31/787 20060101ALI20121105BHEP

17Q First examination report despatched

Effective date: 20140620

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602010045026

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: A61K0038000000

Ipc: A61K0009060000

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

RIC1 Information provided on ipc code assigned before grant

Ipc: A61L 27/52 20060101ALI20170210BHEP

Ipc: A61K 47/34 20170101ALI20170210BHEP

Ipc: A61K 38/39 20060101ALI20170210BHEP

Ipc: A61K 31/7088 20060101ALI20170210BHEP

Ipc: A61K 9/113 20060101ALN20170210BHEP

Ipc: A61L 27/22 20060101ALI20170210BHEP

Ipc: A61K 9/127 20060101ALN20170210BHEP

Ipc: A61L 27/54 20060101ALI20170210BHEP

Ipc: A61K 31/7004 20060101ALI20170210BHEP

Ipc: A61K 31/715 20060101ALI20170210BHEP

Ipc: A61K 9/06 20060101AFI20170210BHEP

Ipc: A61K 31/787 20060101ALI20170210BHEP

Ipc: A61K 38/46 20060101ALI20170210BHEP

INTG Intention to grant announced

Effective date: 20170317

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

RIN1 Information on inventor provided before grant (corrected)

Inventor name: YANG, CHU-YA

Inventor name: DEMING, TIMOTHY, J.

Inventor name: AO, YAN

Inventor name: SONG, BINGBING

Inventor name: SOFRONIEW, MICHAEL, V.

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

Ref country code: AT

Ref legal event code: REF

Ref document number: 925096

Country of ref document: AT

Kind code of ref document: T

Effective date: 20170915

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602010045026

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: SCHMAUDER AND PARTNER AG PATENT- UND MARKENANW, CH

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20170906

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171206

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 925096

Country of ref document: AT

Kind code of ref document: T

Effective date: 20170906

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171207

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171206

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180106

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602010045026

Country of ref document: DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

26N No opposition filed

Effective date: 20180607

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20180228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180218

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180218

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180218

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20200227

Year of fee payment: 11

Ref country code: GB

Payment date: 20200227

Year of fee payment: 11

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20100218

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20200304

Year of fee payment: 11

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170906

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20170906

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20200225

Year of fee payment: 11

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602010045026

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20210218

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210228

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210228

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210218

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210901

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230502